Methods and kits for making double stranded ribonucleic acids

ABSTRACT

Embodiments of the present invention are directed to methods and kits for the generation of double stranded RNA by RNA dependent RNA polymerases. The methods and kits feature exponential generation of such RNAs through simple steps/ The RNA is suitable for interference with expression in cells.

This application is a 35 U.S.C. 371 filing of International ApplicationNo. PCT/US03/040378, filed Dec. 18, 2003, which claims the benefit under35 U.S.C. 119(e) of U.S. Provisional Application No. 60/435,919, filedDec. 20, 2002.

FIELD OF THE INVENTION

The present invention relates to the methods and kits for making doublestranded ribonucleic acids. Double stranded ribonucleic acids haveapplications for blocking or controlling expression of selected genes.This technique for blocking or controlling expression of selected genesusing double stranded ribonucleic acid is referred to as ribonucleicacid interference.

BACKGROUND OF THE INVENTION

As used in the present application the term “RNA” refers to ribonucleicacid and “DNA” refers to a deoxyribonucleic acid. The genetic code iscarried in the sequence of nucleotides comprising the DNA of livingcells. Living cells make proteins by making an RNA copy, or transcript,of portions of such DNA encoding a gene. This RNA, messenger RNA, orsimply mRNA, cases the message of the nature of the protein that will bemade to the cell organelles engaged in protein synthesis. The process ofmaking proteins, moving from the genetic code to the final protein, isreferred to as “expression”.

Double stranded nucleic acid refers to nucleic acid that is paired withits complement through Watson Crick binding. The letters “ds” will beused to denote double stranded nucleic acid. Double stranded ribonucleicacids, dsRNAs, are used for RNA interference, or simply, RNAi. RNAi is aprocess for interfering with the expression of proteins by the cellsengaged in protein synthesis. RNAi has applications for controllingexpression in living cells, for use in cell culture and fermentationprocesses, and in therapies.

RNA interference (RNAi) is one of the oldest and most ubiquitouseukaryotic regulatory mechanisms known and only recently has itsapplication as a research tool become fully realized (Maine, E. M.,2001; Ullu E et al, 2002; Hutvager and Zamore, 2002; Brantl 2002;Lindenbach and Rice, 2002). RNAi is a naturally occurring process inwhich the degradation of gene-specific cellular RNAs results from theintroduction of homologous double-stranded RNAs or “silencer” RNAs(siRNAs). In this way, the expression of specific genes of interest canbe precisely turned off by introducing siRNAs containing sequencesderived from the target cellular RNA. This approach, called reversegenetic analysis, makes it possible to discover the function ofpreviously unknown genes that may play a role in human health. Due toits specificity in gene targeting and compatibility with well-definedcell culture systems, RNAi is the method of choice for studying the vastnumber of available new gene sequences resulting from current genomesequencing projects (Ueda R, 2001). RNAi avoids the need for the costlyand time-consuming process of generating knockout animals, therebylowering the cost of genetic studies and making it possible to studyorganisms previously considered not to be open to genetic analysis.

Present methods of making such nucleic acids are time consuming,awkward, and expensive. Most methods involve generating RNA transcriptsfrom DNA every time material is needed. This requires two separaterounds of synthesis, and a separate hybridization step. The methodrequires a significant amount of DNA template that itself must be madeat regular intervals.

The term “amplify” is used herein in the sense of making more than onecopy. Enzymatic chain reactions, of which the polymerase chain reaction(PCR) is one example, make multiple copies of a nucleic acid having adesired sequence. RNA dependent RNA polymerases use RNA as a template togenerate copies of the template. This document will use the designation“RDRP” for RNA dependent RNA polymerase.

It would be advantageous to generate double-stranded RNAs (dsRNA) foruse as siRNAs in RNAi experiments in a single reaction, with a singleenzyme without further treatment. It would also be advantageous if RNAcould be used as the template for this reaction, thereby alleviating theneed for repeated preparation of DNA templates. These and other benefitsand advantages are obtained with embodiments of the present inventionwhich are summarized in the following section.

SUMMARY OF THE INVENTION

Embodiments of the present invention are directed to methods and kitsfor generating double stranded RNA. One embodiment of the presentinvention is directed to a method of making double stranded RNA having aselected sequence comprising the step of forming an admixture of an RNAdependent RNA polymerase, reagents for the synthesis of transcriptnucleic acids, and at least one template nucleic acid. The templatenucleic acid acts as a template for the synthesis of RNA encoding theselected sequence upon the imposition of nucleic acid synthesisconditions and in the presence of said reagents and RNA dependent RNApolymerase. The method further comprises the step of imposing nucleicacid synthesis conditions on the admixture to form an amplificationproduct comprising double stranded RNA encoding the selected sequence.

Preferably, the template nucleic acid is a deoxyribonucleic acid. And,preferably, RNA dependent RNA polymerase is Q-Beta replicase andmodifications thereto. Other RNA dependent RNA polymerases comprisepolymerases associated with turnip yellow mosaic virus, turnip crinklevirus, tobacco vein mottling virus, and hepatitus C virus, and NS5Bprotein and poliovirus30 pol protein.

The reaction product comprising double stranded RNA has applications forRNAi. That is, the dsRNA formed as an amplification product inhibits theexpression of a selected gene in a cell.

Preferably, the template nucleic acid has portions represented by theformula:5′ A-B-C 3′.At least one letter A and C represents a sequence recognized by the RNAdependent RNA polymerase and at least one of A and C represents theantisense of said sequence recognized by the RNA dependant RNApolymerase. The letter B represents a sequence corresponding to theselected sequence or the antisense of the selected sequence.

The sequence represented in the formula above can be readilysynthesized. That is, the sequence represented by A and C aresynthesized with the sequence represented B. This nucleic acid can becloned into suitable plasmids and other vectors for ease of use. In thealternative the sequence represented by A and C are cloned to thesequence represented by B.

Preferably, the template is a deoxyribonucleic acid. And, the admixturefurther comprises a DNA-dependent RNA polymerase, such as T7 RNApolymerase, SP6, T3, and RNA polymerase 1. The DNA-dependent RNApolymerase is used to transcribe the DNA template to make at least oneRNA recognized by said RNA dependent RNA polymerase. The RNA dependentRNA polymerase generates an amplification product.

Preferably, the reagents for the synthesis of nucleic acid comprisemodified nucleotides. For example, without limitation, preferredmodified nucleotides have modifications at the number two position, suchas 2′-amino, 2′-fluoro, 2′-azido, 2′Omethyl, 2′ ara.

A further embodiment of the present invention is directed to a kit formaking double stranded RNA. The kit comprises an RNA dependent RNApolymerase which synthesizes double stranded nucleic acid in thepresence of reagents and conditions suitable for nucleic acid synthesis.The kit further comprises reagents for the synthesis of transcriptnucleic acids; and means for making at least one template nucleic acid.The template nucleic acid acts as a template for the synthesis of RNAencoding the selected sequence upon the imposition of nucleic acidsynthesis conditions and in the presence of the reagents and RNAdependent RNA polymerase. The kit further comprises instructions forimposing nucleic acid synthesis conditions on said admixture to form anamplification product comprising double stranded RNA encoding theselected sequence. The kit would have individual components packaged ina conventional manner with vials containing reagents, buffers and thelike boxed with instructions.

The double stranded RNA made can preferably be used for RNAi purposes.

Preferably, the means for making at least one template nucleic acid is adeoxyribonucleic acid. This DNA encodes sequences corresponding to theselected sequence.

Preferably, the RNA dependent RNA polymerase is Q-Beta replicase andmodifications thereto. Q-Beta replicase and some modifications of suchenzyme are, under certain conditions, capable of using DNA as atemplate.

Preferably, the template nucleic acid has portions represented by theformula:5′ A-B-C 3′wherein at least one letter A and C represents a sequence recognized bythe RNA dependent RNA polymerase and at least one of A and C representsthe antisense of said sequence recognized by the RNA dependant RNApolymerase The letter B represents a sequence corresponding to theselected sequence or the antisense of the selected sequence.

The sequence represented by A and C can be synthesized with the sequencerepresented B. Or, in the alternative, the sequence represented by A andC are cloned to the sequence represented by B. Cloning and synthesis arefacilitated where the template is a deoxyribonucleic acid. The sequencerepresented by A-B-C can be readily maintained in plasmids.

Preferably, where the template is DNA said admixture further comprises aT7 RNA polymerase. The T7 RNA polymerase transcribes the template tomake at least one RNA recognized by said RNA dependent RNA polymerasewhich RNA dependent RNA polymerase generates an amplification product.

During the making of the amplification product, the RNA formed mayincorporate modified nucleotides. The reagents for the synthesis ofnucleic acid may comprise modified nucleotides for such purpose.Preferably, the modified nucleotides have modifications at the numbertwo position. By way of example without limitation modified nucleotidescomprise limited 2′-amino, 2′-fluoro, 2′-azido, 2′Omethyl, 2′ ara.

Embodiments of the present invention allow the making of largequantities of dsRNA. RNA dependent RNA polymerases generateamplification products exponentially. The process featured in thepresent methods and kits have fewer steps and take shorter times togenerate an amount of dsRNA.

These and other advantages will be apparent to those skilled in the artupon viewing the drawings and reading the brief description of thedrawing, and the detailed description of the drawings which follow.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 depicts a kit embodying features of the present invention;

FIG. 2 depicts a schematic flow diagram of the making of a templatenucleic acid by cloning;

FIG. 3 depicts a schematic flow diagram of the making of a templatenucleic acid by PCR;

FIG. 4 depicts a schematic flow diagram of the direct synthesis of atemplate nucleic acid from a DNA oligonucleotide;

FIG. 5 depicts a schematic flow diagram of the amplification of an RNAtemplate in accordance with features of the present invention and use ofsuch amplification product for RNAi; and

DETAILED DESCRIPTION OF THE INVENTION

RNA interference (RNAi) is one of the oldest and most ubiquitouseukaryotic regulatory mechanisms known and only recently has itsapplication as a research tool become fully realized (Maine, E. M.,2001; Ullu E et al, 2002; Hutvager and Zamore, 2002; Brantl 2002;Lindenbach and Rice, 2002). RNAi is a naturally occurring process inwhich the degradation of gene-specific cellular RNAs results from theintroduction of homologous double-stranded RNAs or “silencer” RNAs(siRNA). In this way, the expression of specific genes of interest canbe precisely turned off by introducing siRNAs containing sequencesderived from the target cellular RNA. This approach, called reversegenetic analysis, makes it possible to discover the function ofpreviously unknown genes that may play a role in human health. Due toits specificity in gene targeting and compatibility with well-definedcell culture systems, RNAi is the method of choice for studying the vastnumber of available new gene sequences resulting from current genomesequencing projects (Ueda R, 2001). RNAi avoids the need for the costlyand time-consuming process of generating knockout animals, therebylowering the cost of genetic studies and making it possible to studyorganisms previously considered not to be open to genetic analysis.

Embodiments of the present invention will be described with respect to akit for making double stranded RNA. A kit embodying features of thepresent invention is depicted in FIG. 1, and is generally designated bythe numeral 11. The kit comprises the following major elements: an RNAdependent RNA polymerase, contained in a suitable containment vessel 15,instructions 19 for the use of such polymerase for making RNAicompositions and packaging 21.

Containment vessels, such as containment vessel 15, may take differentforms. Examples of containment vessels include vials, which areillustrated, envelopes, tins, ampules and the like. The contents of thecontainment vessels may be ready for use or dehydrated forreconstitution.

The enzyme is contained in vial 15, an RNA dependent RNA polymerase,synthesizes double stranded nucleic acid in the presence of reagents.Preferably, the kit 11 further comprises reagents in one or moreadditional containment vessels of which only one is illustrated,designated vial 23, for the synthesis of transcript nucleic acids; andmeans for making at least one template nucleic acid. The templatenucleic acid acts as a template for the synthesis of RNA encoding theselected sequence upon the imposition of nucleic acid synthesisconditions and in the presence of the reagents and RNA dependent RNApolymerase.

The kit 11 further comprises instructions 19 for imposing nucleic acidsynthesis conditions on an admixture of reagents to form anamplification product comprising double stranded RNA encoding theselected sequence. Preferably, the kit would instruct the user as to theuse of the double stranded RNA for controlling expression in cellularprocesses.

The instructions would describe a method of making double stranded RNAhaving a selected sequence comprising the step o forming an admixture ofan RNA dependent RNA polymerase, reagents for the synthesis oftranscript nucleic acids, and at least one template nucleic acid. Thetemplate nucleic acid acts as a template for the synthesis of RNAencoding the selected sequence upon the imposition of nucleic acidsynthesis conditions and in the presence of said reagents and RNAdependent RNA polymerase. The method further comprises the step ofimposing nucleic acid synthesis conditions on the admixture to form anamplification product comprising double stranded RNA encoding theselected sequence.

Preferably, the template nucleic acid has portions represented by theformula:5′ A-B-C 3′wherein the at least one letter A and C represents a sequence recognizedby said RNA dependent RNA polymerase and at least one of said A and Crepresents the antisense of said sequence recognized by said RNAdependant RNA polymerase. The letter B represents a sequencecorresponding to the selected sequence or the antisense of the selectedsequence.

The sequence represented by A and C can be synthesized with the sequencerepresented B. Or, in the alternative, the sequence represented by A andC are cloned to the sequence represented by B. Cloning and synthesis arefacilitated where the template is a deoxyribonucleic acid. The sequencerepresented by A-B-C can be readily maintained in plasmids.

Preferably, where the template is DNA said admixture further comprises aT7 RNA polymerase. The T7 RNA polymerase transcribes the template tomake at least one RNA recognized by said RNA dependent RNA polymerasewhich RNA dependent RNA polymerase generates an amplification product.The kit may further comprise the enzyme T7 RNA polymerase in the vial 15or vial 23, or in a separate vial (not shown).

The kit 11 would have individual components packaged in a conventionalmanner, with vials 15 and 23, containing enzymes, reagents, buffers andthe like boxed with instructions, in packaging 21. Packaging 21 is inthe form of a box, but may take many forms including bags, wraps,bundles plastic formed containers and the like common in the industry.

The double stranded RNA made with kit 11 can preferably be used for RNAipurposes. Preferably, the instructions comprise directions for the RNAipurposes.

Embodiments of the present invention feature an enzyme, Q-Betareplicase, a powerful replicator of RNAs providing 10¹³ foldamplification within 30 minutes at room temperature. Q-beta replicase isa well-studied enzyme often studied for its robust RNA amplificationproperties (Biebricher C K, Eigen M, Gardiner W C Jr. Kinetics of RNAreplication. Biochemistry. 1983 May 10; 22(10):2544-59). One of theproducts of Q-beta replication is double-stranded RNA (Biebricher, C K,Eigen, M, and Gardiner, W C. (1984) Kinetics of RNA replication:plus-minus asymmetry and double-strand formation. Biochemistry, 23,3186-3194; Axelrod V D, Brown E, Priano C, Mills D R. Coliphage Q betaRNA replication: RNA catalytic for single-strand release. Virology; 1991October; 184(2):595-608).

Protocals for the isolation and or manufacture of the enzyme are knownin the art. A preferred enzyme is sold under the name of Q-Amp™ byQ-RNA, Inc. This enzyme has been modified to improve the manner in whichit is isolated and purified. Preferably, the enzyme has no contaminationfrom cellular RNAs endogenous to bacteria and has no detectable nucleaseactivities.

Q-Beta replicase amplification reactions are simple to set up, require asingle tube and do not require special renaturation procedures. By wayof example, without limitation, one set of conditions for formingamplification products with the enzyme Q-Beta replicase is 200 microMNTPs, 80 milliM TRIS-HCl, pH 7.5, 10 milliM MgCl₂, 2 milliM DTT. Thereaction tolerates a wide range of salt conditions (eg. 0 to 500 milliMNaCl), can be initiated with small amounts of template (1 nanogram downto a single molecule) and Q-beta replicase may be present at aconcentration of 50-200 nanoM.

Q-Beta replicase can incorporate modified nucleotides, enabling thesynthesis of stabilized RNAs or RNAs with other functional groups. Undercertain conditions Q-Beta replicase can use DNA as a template. Such useof DNA as a template can further simplify the generation of dsRNAs. Useof Q-Beta replicase is a significant advance over current techniques.

There are currently on the market kits for the express purpose ofgenerating siRNA for RNAi experiments. Before using the kit, aresearcher must first plan and design a few potential siRNA sequencesand then order these sequences encoded in several syntheticoligonucleotides. The oligonucleotides must be assembled as templatesfor T7 transcription, followed by transcription reactions in separatetubes. The products of these reactions are then combined and annealed toform siRNAs. Although one synthesis should yield enough RNA for severalreactions, eventually the procedure must be repeated in its entiretywhen the RNA stock runs out.

With Q-Beta replicase, a single transcription can generate aninexhaustible supply of RNA template. Only 1 pg or less is needed toinitiate a reaction that can generate microgram amounts of dsRNA.Because both strands can be made in the same reaction vessel, there isno need for separate transcription reactions or additional annealingsteps—the RNAs will anneal automatically as their concentrationsincrease. Therefore high yields of dsRNA can be generated in underthirty minutes.

It is also possible to combine T7 RNA polymerase and Q-Beta replicase ina single reaction. The simplest approach for generating siRNAs withRDRPs is to use DNA directly as a template. Although the efficiency ofDNA as a template is about 1000-fold lower than RNA in some cases allthat need be generated are enough RNA transcripts that can themselves beused as high efficiency templates. Again, all of these steps can occurin a single tube. Finally, an additional benefit of using RDRPs is thatthe products can also serve as templates. Therefore future rounds of invitro transcription are no longer necessary—the reaction products fromprevious rounds of siRNA synthesis can be used as templates, therebycreating a virtually inexhaustible supply of template.

RNA can than be diluted and a single picogram would be used to seed thereaction. So 1 ug of synthesized RNA is enough for 10⁶ siRNA reactionswith a RDRP. Because of the nature of RDRPs, there is never a need torepeat the initial RNA transcription because a RDRP can use its ownamplification product as a template. Thus only a single synthesis of RNAtemplate must be prepared for virtually a lifetime supply of template togenerate dsRNA.

RDRP also have a lower level of activity on DNA templates. Our researchshows that the sensitivity of amplification of DNA is roughly 1000-foldlower than with RNA as a template. Therefore if 1 picogram of RNAtemplate is required to seed an amplification reaction, 1 ng of DNA willbe needed on average. A typical synthesis of DNA oligos at the 250 nmolescale, the smallest scale commercially available, will yield hundreds ofmicrograms, which relates to thousands of amplification reactions. Inessence, the DNA template becomes obsolete once enough RNA template isgenerated due to the higher efficiency of amplification. Therefore, oneoligonucleotide synthesis per siRNA is all that is required in terms ofconstruction of the siRNA template.

Use of the double stranded product as a siRNA will follow currentlyacceptable techniques. There are currently several kits on the marketthat can enable the transfection of siRNAs into cells. Our approachwould take advantage of these pre-existing methods. There are twogeneral approaches in the synthesis of siRNAs. One is to rationallytarget specific regions in a cellular RNA and to generate small RNAs tothese regions. Typically several such constructs are made targetingdifferent regions in a cellular RNA in hopes that at least one will showacceptable efficiency in terminating the expression of the target gene.Another approach is to generate a long dsRNA containing much of thecellular RNA. Such RNAs can either be introduced to cells, which processthe dsRNA into ˜22 nucleotide siRNAs (Elbashir, S. M., Lendeckel, W.,and Tuschl, T. 2000. RNA interference is mediated by 21- and22-nucleotide RNAs. Genes and Development, 15:188-200). Another approachis to process the long dsRNA into smaller pieces before transfectioninto cells by digesting with a double stranded ribonuclease like Dicer(Grosshans, H and Slack, F J. 2002. The Journal of Cell Biology, 156(1):17-21), E. coli RNase III, or other RNase III-like double-strandedribonucleases (Lamontagne B et al. 2001. Curr Issues Mol. Biol.3(4):71-8). dsRNAs generated by RDRPs are suitable for all of thesetechniques.

Further advantages of the present invention will be apparent from thedescription provided in the following Examples.

EXAMPLE NUMBER 1 Making a Template

Example 1 describes the making of a template with reference to FIG. 2.FIG. 2 depicts a flow diagram representing steps in a process for makinga template for use in making double stranded RNA.

Each step is performed in accordance with standard techniques known toindividuals skilled in the art. FIG. 2 illustrates a plasmid withsuitable restriction sites and a T7 promoter site. A nucleic acid havingthe formula:5′ A-B-C 3′.At least one letter A and C represents a sequence recognized by an RNAdependent RNA polymerase and at least one of said A and C represents theantisense of the sequence recognized by the RNA dependant RNA polymeraseis cloned into the multi-cloning site. The letter B represents asequence corresponding to the selected sequence or the antisense of theselected sequence.

Next, the plasmid in expressed in a suitable vector and purified. Theplasmid in linearized with SmaI. The linearized nucleic acid is thentranscribed to generate a template that is recognized by the RNAdependant RNA polymerase. As used herein the word “recognized” meansthat the enzyme uses such nucleic acid to generate further amplificationproduct, either the plus or minus strand, of the template.

EXAMPLE 2 Making a Template with PCR Processes

This Example 2 will describe the making of a template with PCRprocesses. For short templates, synthetic oligonucleotides may decreasethe time of construction. With reference to FIG. 3, a DNA molecule issynthesized. A nucleic acid is synthesized having the formula, or itsantisense:5′ A-B-C 3′At least one letter A and C represents a sequence recognized by an RNAdependent RNA polymerase and at least one of said A and C represents theantisense of the sequence recognized by the RNA dependant RNA polymeraseis cloned. The letter B represents a sequence corresponding to theselected sequence or the antisense of the selected sequence. Thissequence is denoted “siRNA” in the Figure.

The nucleic acid is amplified by PCR using primers which incorporate aT7 promoter sequence. The amplified DNA is transcribed with T7polymerase to generate a transcription product. The transcriptionproduct is amplified with an RNA dependant RNA polymerase such as Q-Betareplicase.

EXAMPLE 3 Direct Synthesis of Template Nucleic Acid from DNAOligonucleotide

This Example 3 describes the direct synthesis of double stranded RNAfrom a DNA oligonucleotide. Under certain conditions RNA dependant RNApolymerases may read DNA templates directly.

With reference to FIG. 4, a DNA molecule is synthesized having theformula:5′ A-B-C 3′At least one letter A and C represents a sequence recognized by an RNAdependent RNA polymerase and at least one of said A and C represents theantisense of the sequence recognized by the RNA dependant RNA polymeraseis cloned. The letter B represents a sequence corresponding to theselected sequence or the antisense of the selected sequence. Thissequence is denoted “siRNA” in the Figure.

The nucleic acid is amplified by Q-Beta replicase reading the DNAtemplate directly. Amplification of DNA templates are similar to theamplification of RNA templates and by way of example, withoutlimitation, one set of conditions for forming amplification products is50-200 nanoM Q-Beta replicase, 200 microM NTPs, 80 milliM TRIS-HCl, pH7.5, 10 milliM MgCl₂, 2 milliM DTT. The amount of DNA required toinitiate amplification by Q-beta replicase is typically 100 to1000-times more than its RNA counterpart. Therefore, at least 10⁷strands of DNA should suffice for any template. After amplification, DNAtemplates can efficiently be removed from the reaction bydeoxyribonucleases such as DnaseI.

EXAMPLE 4 Amplification of RNA by RNA Dependant RNA Polymerase

The RNA products of Examples 1-3 are amplifiable by RNA dependant RNApolymerases. One set of conditions for forming amplification productswith the enzyme Q-Beta replicase is 100 nanoM Q-Beta replicase, 200microM NTPs, 80 milliM TRIS-HCl, pH 7.5, 10 milliM MgCl₂, 2 milliM DTT.Conditions for amplification with other enzymes are as reported in theliterature.

Due to manner in which RNA dependant RNA polymerases generate nucleicacid, the synthesis of template nucleic acid occurs exponentially. Theenzymes recognize both the sense and antisense strands as templates. Asingle molecule can be amplified to a visible product in twenty minutesat room temperature.

EXAMPLE 5 Use of Double Stranded RNA Derived from Amplification for RNAiPurposes

FIG. 5 depicts the amplification of RNA and its use in cells to controltranslation processes. RNA produced by amplification by the RNAdependant RNA polymerase is used for RNAi purposes as reported in theliterature.

Thus, embodiments of the present invention have been described withrespect to the examples, figures, and the detailed description containedherein with the understanding that individuals skilled in the art areable to make modifications and alterations to the present teaching.Therefore, the invention should not be limited to the specifics of suchexamples, figures and detailed description but should encompass the fullscope of the subject matter of the following claims.

REFERENCES

-   Axelrod V D, Brown E, Priano C, Mills D R. Coliphage Q beta RNA    replication: RNA catalytic for single-strand release. Virology. 1991    October; 184(2):595-608.-   Biebricher C K, Eigen M, Gardiner W C Jr. Kinetics of RNA    replication. Biochemistry. 1983 May 10; 22(10):2544-59.-   Brantl S. (2002) Antisense-RNA regulation and RNA interference.    Biochim Biophys Acta. 3; 1575(1-3):15-25.-   Elbashir, S. M., Lendeckel, W., and Tuschl, T. 2000. RNA    interference is mediated by 21- and 22-nucleotide RNAs. Genes and    Development, 15:188-200.-   Hutvagner G, Zamore P D. (2002) A MicroRNA in a Multiple-Turnover    RNAi Enzyme Complex. Science [epub ahead of print].-   Lamontagne B, Larose S, Boulanger J, Elela S A. (2002) The RNase III    family: a conserved structure and expanding functions in eukaryotic    dsRNA metabolism. Curr Issues Mol. Biol. 3(4):71-8.-   Lindenbach B D, and Rice C M. (2002) RNAi targeting an animal virus:    news from the front. Mol Cell. 9(5):925-7.-   Maine, E. M. (2001) RNAi As a tool for understanding germline    development in Caenorhabditis elegans: uses and cautions. Dev Biol    239; 177-89.-   Ueda R. (2001). Rnai: a new technology in the post-genomic    sequencing era. J Neurogenet. 15(3-4):193-204.-   Ullu E, Djikeng A, Shi H, and Tschudi C. (2002) RNA interference:    advances and questions. Philos Trans R Soc Lond B Biol Sci. 29;    357(1417):65-70.

1. A method of making double stranded RNA having a selected sequencecomprising the steps of: a) forming an admixture of an RNA dependent RNApolymerase, reagents for the synthesis of transcript nucleic acids, andat least one template nucleic acid, said template nucleic acid acting asa template for the synthesis of RNA encoding said selected sequence uponthe imposition of nucleic acid synthesis conditions and in the presenceof said reagents and RNA dependent RNA polymerase; b) imposing nucleicacid synthesis conditions on said admixture to form an amplificationproduct comprising double stranded RNA encoding the selected sequence.2. The method of claim 1 wherein said template nucleic acid is adeoxyribonucleic acid.
 3. The method of claim 1 wherein said RNAdependent RNA polymerase is Q-Beta replicase and modifications thereto.4. The method of claim 1 wherein said reaction product comprising doublestranded RNA inhibits the expression of a selected gene in a cell. 5.The method of claim 1 wherein said template nucleic acid has portionsrepresented by the formula:5′ A-B-C 3′ wherein at least one letter A and C represents a sequencerecognized by said RNA dependent RNA polymerase and at least one of saidA and C represents the antisense of said sequence recognized by said RNAdependant RNA polymerase, and the letter B represents a sequencecorresponding to the selected sequence of the antisense of said selectedsequence.
 6. The method of claim 5 wherein said sequence represented byA and C are synthesized with the sequence represented B.
 7. The methodof claim 5 wherein said sequence represented by A and C are cloned tothe sequence represented by B.
 8. The method of claim 5 wherein saidtemplate is a deoxyribonucleic acid.
 9. The method of claim 8 whereinsaid admixture further comprises a DNA-dependent RNA polymerase, said T7DNA-dependent RNA polymerase transcribing said template to make at leastone RNA recognized by said RNA dependent RNA polymerase which RNAdependent RNA polymerase generates an amplification product.
 10. Themethod of claim 1 wherein said reagents for the synthesis of nucleicacid comprise modified nucleotides.
 11. The method of claim 10 whereinsaid modified nucleotides have modifications at the number two position.12. The method of claim 11 wherein said modified nucleotides compriselimited 2′-amino, 2′-fluoro, 2′-azido, 2′Omethyl, 2′ ara.
 13. The methodof claim 1 wherein said amplification product is used for RNAi.
 14. Akit for making double stranded RNA comprising: a) an RNA dependent RNApolymerase which RNA dependent RNA polymerase synthesizes doublestranded nucleic acid in the presence of reagents; b) reagents for thesynthesis of transcript nucleic acids; c) means for making at least onetemplate nucleic acid, said template nucleic acid acting as a templatefor the synthesis of RNA encoding said selected sequence upon theimposition of nucleic acid synthesis conditions and in the presence ofsaid reagents and RNA dependent RNA polymerase; d) instructions forimposing nucleic acid synthesis conditions on said admixture to form anamplification product comprising double stranded RNA encoding theselected sequence.
 15. The kit of claim 14 wherein said template nucleicacid is a deoxyribonucleic acid.
 16. The kit of claim 14 wherein saidRNA dependent RNA polymerase is Q-Beta replicase and modificationsthereto.
 17. The kit of claim 14 wherein said reaction productcomprising double stranded RNA inhibits the expression of a selectedgene in a cell.
 18. The kit of claim 14 wherein said template nucleicacid has portions represented by the formula:5′ A-B-C 3′ wherein at least one letter A and C represents a sequencerecognized by said RNA dependent RNA polymerase and at least one of saidA and C represents the antisense of said sequence recognised by said RNAdependant RNA polymerase, and the letter B represents a sequencecorresponding to the selected sequence of the antisense of said selectedsequence.
 19. The method of claim 18 wherein said sequence representedby A and C are synthesized with the sequence represented B.
 20. The kitof claim 18 wherein said sequence represented by A and C are cloned tothe sequence represented by B.
 21. The kit of claim 18 wherein saidtemplate is a deoxyribonucleic acid.
 22. The kit of claim 18 whereinsaid admixture further comprises a DNA-dependent RNA polymerase, saidDNA-dependent RNA polymerase transcribing said template to make at leastone RNA recognized by said RNA dependent RNA polymerase which RNAdependent RNA polymerase generates an amplification product.
 23. The kitof claim 14 wherein said reagents for the synthesis of nucleic acidcomprise modified nucleotides.
 24. The kit of claim 23 wherein saidmodified nucleotides have modifications at the number two position. 25.The kit of claim 24 wherein said modified nucleotides comprise limited2′-amino, 2′-fluoro, 2′-azido, 2′Omethyl, 2′ ara.
 26. The kit of claim14 wherein said amplification product is used for RNAi.